Oral immune dysfunction is associated with the expansion of FOXP3+PD-1+Amphiregulin+ T cells during HIV infection

Residual systemic inflammation and mucosal immune dysfunction persist in people living with HIV, despite treatment with combined anti-retroviral therapy, but the underlying immune mechanisms are poorly understood. Here we report that the altered immune landscape of the oral mucosa of HIV-positive patients on therapy involves increased TLR and inflammasome signaling, localized CD4+ T cell hyperactivation, and, counterintuitively, enrichment of FOXP3+ T cells. HIV infection of oral tonsil cultures in vitro causes an increase in FOXP3+ T cells expressing PD-1, IFN-γ, Amphiregulin and IL-10. These cells persist even in the presence of anti-retroviral drugs, and further expand when stimulated by TLR2 ligands and IL-1β. Mechanistically, IL-1β upregulates PD-1 expression via AKT signaling, and PD-1 stabilizes FOXP3 and Amphiregulin through a mechanism involving asparaginyl endopeptidase, resulting in FOXP3+ cells that are incapable of suppressing CD4+ T cells in vitro. The FOXP3+ T cells that are abundant in HIV-positive patients are phenotypically similar to the in vitro cultured, HIV-responsive FOXP3+ T cells, and their presence strongly correlates with CD4+ T cell hyper-activation. This suggests that FOXP3+ T cell dysregulation might play a role in the mucosal immune dysfunction of HIV patients on therapy.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability RNA sequencing data from healthy human participants that support the findings of this study have been deposited in GEO, NCBI with the GSE167211 accession code. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167211. Transcriptome data from HIV+ patients are deposited at the NCBI Genotypes and Phenotypes (dbGaP)data repository. These data are open to general research use (dbGaP Study Accession: phs002364.v1.p1) and available at https:// www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs002364.v1.p1. Other data that support the findings of this study are also available from the corresponding author upon reasonable request.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Power analyses was done based on estimates. For the power and sample size calculation, we used https://www.stat.ubc.ca/~rollin/stats/ ssize/n2.html We used relevant population values for mu1 (mean of population 1), mu2 (mean of population 2), and sigma value of 0.5 (common standard deviation) 0.05 as α (type I error rate) and a 2 sided test for power calculation. Because our preliminary data showed that HIV+ group had atleast 1.4 fold increase in Treg proportions, we used 1 and 1.4 as mu1 and mu2 respectively. Our desired power was set to 0.8.
Data exclusions Data points were not excluded.

Replication
We performed at least triplicate repeats of the in vitro experiments with independent biological replicates in each experiment. All replicate experiments showed reproducibly similar data and were successful.
Randomization Human participants were randomly assigned in control and HIV+ groups, with representation of males and females in each group. All other in vitro experiments were also performed using randomly allocated tonsils.

Blinding
ELISAs were performed by a technician who was blinded to the identity of the saliva and supernatant samples. The investigators were also blinded to group allocation during RNA-seq data collection and/or analysis. It was not possible to do complete blinding for the in vitro and flow cytometry experiments, as the same research associate performed the cell culture and the staining. However, the person who did the flow-cytometry data analysis was blinded on the groups until the final analysis of all the replicate experiments at which point the research associate released the codes for the cell-culture groups.
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Validation
All antibodies were commercially available, validated and were used in previous studies, as per the references available in manufacturer's websites. For the BD-Biosciences flow cytometry antibodies, the company website says "The production process underwent stringent testing and validation to assure that it generates a high-quality conjugate with consistent performance and specific binding activity". The invitrogen/Thermofisher website says: "To help ensure superior antibody results, we've expanded our specificity testing methodology using a 2-part approach for advanced verification".
We also titrated the antibodies using unstained or Isotype controls prior to use.

Human research participants
Policy information about studies involving human research participants

Population characteristics
Informed consents from healthy individuals and Cleveland HIV+ cohort under a protocol approved by the University Hospitals the Cleveland Medical Center Institutional Review Board. n= 78, Males= 49, Females = 29; Healthy control subjects were at least 18 years of age and in good general health (Table.1). HIV+ participants were 18 years or older, and were HIV positive with cART treatment for at least 1 year. > 75 % of HIV+ patients reported prior and current soft tissue lesions, gingivitis, and periodontitis. For the periodontitis study, healthy controls (n=8; 5 females and 3 males) and periodontitis patients (Perio. n= 9; 6 females and 3 males)were recruited under a separate UH-IRB protocol. Demographics was consistent with the general population in the Cleveland-Akron metro area.

Recruitment
Participants were recruited after obtaining informed consents. Healthy control subjects were at least 18 years of age and in good general health. Exclusion criteria were oral inflammatory lesions (including gingivitis and periodontitis), oral cancer diagnosis, soft tissue lesions, and the use of tobacco in the past month. HIV+ participants were 18 years or older, and were HIV positive with cART treatment for at least 1 year. Exclusion criteria were oral cancer diagnosis and the use of tobacco in the past month. The inclusion and exclusion criteria were the same for periodontitis study, except that the the inclusion criteria for the periodontitis group included the presence of periodontitis.

nature research | reporting summary
April 2020 Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation For single-cell flow cytometry staining, cells isolated and processed ex vivo from tissues or lymphoid organs as well as cultured cells were washed in PBS or PBS/BSA, and blocked by Fc receptor blocking, before surface staining using the antibodies. For Foxp3 and other intracellular marker staining, the cells were fixed with Foxp3 fix-perm set (eBioSciences/ Thermofisher) after surface staining. Live-Dead viability staining was used to remove dead cells in the analyses. Appropriate un-stain, isotype, secondary antibody, single stain and FMO controls were used and representative data are shown in supplementary figures. Before intracellular cytokine staining, cultures were re-stimulated with PMA (50 ng/ml) and Ionomycin (500 ng/ml) for 4 hours, with brefeldin-A (10 μg/ml) added in last 2 hours. For phospho staining, the cells were washed, fixed and were stained with Phosflow staining kit from BD Biosciences using manufacturer's protocol.

Instrument BD Fortessa
Software Flowjo versions 9.8,9.9.6,10.5.3,and 10.7.1 Cell population abundance CD4+ T ells were more abundant in PBMC and tonsils than in the oral tissues.

Gating strategy
Gating strategy: Preliminary FSC/SSC gates for the starting leukocyte cell population, and subsequent gating to include singlets, and CD3+ T cells were used. Boundaries of the "positive" gates were assigned based on the unstained controls, PBMC/d0 negative controls, and FMO controls. We have shown data exemplifying the gating strategies and the controls where appropriate in figures S1B, S1C, S5B, S5D, S8, and S19 in the supplementary Information.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.